Protocol to Reprogram Human Menstrual Blood-Derived Stromal Cells to Generate AOX15-iPSCs.

Cell reprogramming has revolutionized the fields of cell and regenerative biology. However, human induced pluripotent stem cell (iPSC) derivation remains inefficient and variable. Here, we present a protocol that uses human menstrual blood-derived stromal cells (MnSCs), which are susceptible to reprogramming, as a source of somatic cells. We describe an oocyte-based reprogramming combination to generate AOX15-iPSCs that can be used to study different states of pluripotency. For complete details on the use and execution of this protocol, please refer to Lopez-Caraballo et al. (2020).

Before You Begin

Prepare the media below. Pre-warm the media intended for cell culture at 37°C at least 30 min prior to beginning each section of this protocol. Refer to the Key Resources Table for a complete list of materials.

Human Menstrual derived stromal cells (MnSCs) culture Medium: Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% FBS, 1x Glutamax, 1× non-essential amino acids (NEAA) and 1% penicillin/streptomycin (P/S).

Pluripotent cells culture Medium (hES): Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 20% KnockOut Serum Replacement (KSR), 1× Glutamax, 1× non-essential amino acids (NEAA), 1% penicillin/streptomycin (P/S), 50 μM-mercaptoethanol and 8 ng/mL bFGF.

Mouse embryonic fibroblast (MEF) and 293T culture Medium: Dulbecco’s Modification of Eagle Medium (DMEM) supplemented with 10% FBS, 1× non-essential amino acids (NEAA) and 1% penicillin/streptomycin (P/S).

All cell types should be cultured in an incubator at 37°C, 5% CO2, 85% humidity.

MEF inactivation medium: 9.5 mL of MEF medium + 0.5 mL mitomycin stock solution (200 μg/mL). Prepare fresh before use.

Menstrual blood collection medium: 5 mL of calcium/magnesium-free PBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 5 mM EDTA. Keep at 4°C.

MnSC freezing media: FBS with 10% DMSO. Prepare fresh before use. Keep at 4°C.

Preparation of Transfection Reagent PEI

Timing: 2 h

Warm 10 mL of MilliQ water to 80°C.

Dissolve 10 mg of PEI into pre-warmed MilliQ water.

Let the solution reach 25°C.

Measure pH and adjust to pH 7.

Filter solution using 0.22 μm syringe filter.

Aliquot into 1 mL sterile Eppendorf tubes and freeze (−20°C) until use (up to 1 year).

CRITICAL: It is important to adjust pH to exactly 7, higher or lower pH will lead to poor transfection efficiency. Do not perform more than two freeze-thaw cycles.

Mouse Embryonic Fibroblasts (MEF) Thawing, Culturing, and Inactivation

Timing: 4 days

Day 0. MEF thawing.

Thaw one frozen vial of murine embryonic fibroblasts (MEFs)( ≈ 1–3 × 106 cells) in a 37°C water bath. MEFs may be obtained from a number of qualified vendors, see Key Resources Table for specific strain used in this protocol.

Transfer the content of the vial into a 15 mL tube containing 10 mL of MEF medium.

Centrifuge at 700 × g for 4 min to pellet cells.

Remove supernatant.

Resuspend cell pellet in 1 mL of MEF medium using a p1000 micropipette to a single cell suspension pipetting up and down 3–5 times.

Add 14 mL of MEF medium.

Transfer the cell suspension into one 75 cm2 flask (T-75).

Place the MEFs in an incubator at 37°C, 5% CO2, 86% humidity.

CRITICAL: Every frozen MEF preparation thaws a little differently. It is important for the MEFs to be thawed and maintained at a relatively high density (over 50% confluent on day 1 after thawing and after passaging).

Day 2–4 (or when cells reach 100% confluency). Perform MEF inactivation to generate inactivated MEF (iMEF).

Remove the growth medium from the flask and replace it with 10 mL per T75 of freshly prepared inactivation medium (MEF media [9.5 mL] + 0.5 mL mitomycin stock solution [200 μg/mL]) to cover the monolayer.

Place the flask in the incubator (37°C, 5% CO2, 86% humidity) for 3 h.

Aspirate inactivation medium from the flask.

Wash the monolayer of cells twice with 10 mL of DPBS.

Dissociate cells with 0.05% trypsin/EDTA for 5 min.

Add 5 mL of MEF medium and break up cell aggregates by pipetting up and down with a 5 mL serological pipette.

Count cells and dilute to 3.0 × 105 cells/mL in MEF medium.

Plate 1.0 × 105 cells/cm2. (For example, for a 6-well plate we will plate 150,000–200,000 iMEF/well).

Incubate for 12–16 h and use the cells as feeder layers the next day. Feeders can be used up to 5 days after preparation. Renew with fresh MEF medium every other day.

Key Resources Table

Step-By-Step Method Details

Isolation of Menstrual Blood-Derived Stromal Cells (MnSCs)

Timing: 5–10 days

MnSCs are obtained from the menstrual blood of volunteers at the peak of flow (first or second day of menses). The samples are collected in 5 mL of calcium/magnesium-free PBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 5 mM EDTA (collection medium). The cells are harvested with the informed consent of the donor as approved by an institutional review board. All cell lines must be regularly tested to ensure that they are free of mycoplasma prior to reprogramming and every 5–10 passages (we use a PCR-based test, see Key Resources Table for specific reagent used in this protocol).

Prepare 15 mL tubes containing 3 mL of Ficoll (Histopaque 1077-Sigma) and bring to 25°C.

Carefully layer the menstrual blood sample onto the Ficoll Histopaque-1077 (Figures 1A–1C).

Figure 1

Transfer of the Menstrual Blood Sample onto the Ficoll Histopaque-1077

(A) 15 mL tube containing 3 mL of Ficoll Histopaque-1077.

(B and C) Menstrual blood sample layering onto Ficoll avoiding the disturbance of phases.

(D) Supernatant containing MnSCs after centrifugation of menstrual blood sample.

CRITICAL: When transferring the menstrual blood sample into Ficoll-containing tubes it is important to add the blood drop by drop on the tube wall to avoid mixing with the Ficoll phase.

Centrifuge at 400 × g for 30 min at 25°C to pellet red blood cells.

Recover supernatant containing MnSCs (Figure 1D) and transfer into a clean 50 mL tube.

Wash the cells by adding 10 mL of DPBS with 1% penicillin/streptomycin (P/S) and mix by gently drawing in and out of a 10 mL pipette.

Centrifuge at 250 × g for 10 min to pellet stromal cells.

Discard supernatant.

Repeat steps 5–7 twice.

Resuspend cell pellet with 1 mL of MnSC medium using a p1000 micropipette.

Add 6 mL of MnSC and transfer cell suspension into a T25 cell culture flask.

Place in a 5% CO2, 37°C incubator.

Change MnSC medium every other day.

When the cells reach 80%–90% confluency (usually after 5–7 days) (Figure 2) (see Troubleshooting 1), dissociate them by adding 1 mL of 0.05% trypsin-EDTA to the T25 flask after PBS washing and incubate for 5–10 min at 37°C.

Figure 2

MnSC Morphology

Bright-field microscopic image showing isolated MnSC morphology and appearance at 80% confluency. Scale bar, 20 μm.

Add 5 mL of MnSC medium to inactivate the trypsin and mix well by gently pipetting up and down using a 5 mL pipette and plate at 104 cells/cm2.

Split the cells every four – five days to maintain 80% confluence. See Troubleshooting 2 and 3.

CRITICAL: Do not dilute cells below 5 × 103 cells/cm2. Low confluency yields into poor cell proliferation and early senescence.

Pause Point: Cells may be stored at −80°C for short-term storage or in liquid nitrogen for long-term storage. Freeze cells in 10% DMSO/FBS.

Preparation of the Reprogramming Viruses

Timing: 5–8 days

Retroviral particles are prepared from pMX-backbone based vectors (from Cell Biolabs, find information in Key Resources Table) encoding reprogramming factors ASF1A, SOX15 and OCT4 (AOX15)(please find pMX-ASF1A, pMX-SOX15 and pMX-OCT4 vector description in Lopez-Caraballo et al., 2020)(Figure 3). Retroviral particles will be used to over express AOX15 combination in MnSCs.

Figure 3

Schematic Representation of pMXs-Based Vectors

(A and B) Bicistronic retroviral vector that allows expression of human ASF1A or SOX15 and GFP protein, and (C) monocistronic pMXs-based retroviral vector for the expression of human OCT4.

Seed ~5 × 106 cells of HEK293T cells onto a 100 mm tissue culture plate and culture for 24–48 h in 293T medium.

Day 0: When the cells reach 100% confluency split cells at a 1:4 ratio. Allow cells to reach 80%–90% confluency (Figure 4). See Troubleshooting 4 and 5.

Figure 4

293T Morphology

Bright-field image of (A) 100% confluent and (B) 80%–90% confluent 293T cells. Cells should appear similar to image (B) immediately prior to transduction. Scale bar, 50 μm.

Day 1: Remove medium and replace with 10 mL fresh pre-warmed growth medium 1–2 h before transfection.

CRITICAL: Be careful not to dislodge the cells when adding the medium.

Prepare the two solutions listed below in 15 mL Eppendorf tubes for each of the retroviruses:

Add 60 μL PEI (1 μg/μL in water, pH7) to the mixture (4:1 v/w ratio of PEI:DNA), drop by drop while vortexing at medium speed (Methods Video S1).

CRITICAL: When adding PEI to the DNA mix, it is important to add it drop by drop while vortexing.

Vortex briefly (30 s) and leave for 10 min at 25°C.

Add the mixture dropwise in a circular motion, to the 293T cells.

Gently rock the plate in a back and forth and side-to-side motion to ensure even distribution of the transfection mixture. Incubate the plate in a 37°C, 5% CO2 tissue culture incubator for 8 h.

After 8 h aspirate the medium and add 6 mL of 293T medium.

CRITICAL: Do not exceed 12 h post-transfection.

Virus Collection Point 1: Approximately 36 h after transfection (on day 3), collect the supernatant using a disposable syringe and filter the supernatant using a 0.45 μm syringe filter, into a 50 mL conical tube. Use this supernatant for cell transduction or store this tube for 17–18 h at 4°C. For short term storage freeze supernatants down at −20°C (up to 1 month) or at −80°C for long-term storage (up to 6 months).

Gently and slowly add 6 mL of fresh 293T medium down the side of the culture plate and incubate in a 37°C, 5% CO2 tissue culture incubator for 24 h.

CRITICAL: Be careful not to dislodge the cells when removing or adding the medium.

Virus Collection Point 2: On day 4, repeat step one from day 3, by filtering the additional 8 mL of medium into a 50 mL conical tube and use for cell transduction or store as in virus collection point 1.

Virus titration using 293T transduction and flow cytometric analysis:

The day before transduction (day 0), plate 6 × 105 293T cells in a 6-well plate (105 cells/well).

On day 1, add 1 mL or 3 mL of pMX-SOX15, pMX-ASF1A or pMX-OCT4 viral supernatant to each well (6 total). pMX-SOX15 and pMX-ASF1A will allow expression of GFP as they are bicistronic vectors, while pMX-OCT4 will not express GFP (Figure 3) and thus it will be the GFP negative control.

Incubate in a 37°C, 5% CO2 tissue culture incubator for 24 h.

On day 2, replace medium with 2 mL/well of 293T medium.

Incubate for 72 h (replacing the medium 48 h after transduction, on day 4).

On day 5, dissociate transduced and control (non-transduced) 293T cells with 0.05% Trypsin/EDTA, and resuspend pellet with 1 mL of 4% Paraformaldehyde (PFA).

Incubate 20 min at 25°C to fix the cells.

Wash cells adding 10 mL of DPBS, centrifuge at 1,000 × g for 5 min. Repeat this step once again.

Resuspend cells with 0.5–1 mL PBS-0.1% BSA-150 mM EDTA-0.02% sodium azide and analyze cells by flow cytometry.

Pause Point: or store cells at 4°C for up to 1 week.

Analyze GFP positive cells by flow cytometry (Figure 5).

Figure 5

Viral Particles Titration

Flow cytometric analysis of GFP expression of 293T cells transduced with 1 and 3 mL of each pMX-SOX15 and pMX-ASF1A produced viral supernatant. pMX-OCT4 transduced 293T cells are used as GFP negative control (293T Control). Dot plots (A) and histograms (B) showing percentages of GFP positive cells under each condition.

Only viral supernatant reaching an efficiency of 80% GFP positive cells in 1 mL supernatant (and over 95% in 3 mL) should be used for cell reprogramming.

AOX15 Reprogramming of MnSCs

Timing: 3–4 weeks

MnSCs are reprogrammed into iPSC using the oocyte-based factor combination ASF1A, SOX15 and OCT4 (AOX15). Transduction of the reprogramming factors must be carried out using low passage MnSC cells (ideally passage 3–5).

The day before reprogramming (day 0) plate 10 5 MnSCs into 35 mm cell culture plates.

24 h later (day 1), add 5 mL of each pMX-SO15, pMX-ASF1A and pMX-OCT4 retroviral supernatant (it should be a MOI= 5 for each vector), and 15 μL of 8 μg/mL polybrene per plate and return plate to incubator.

When using frozen viral supernatant, thaw them on ice.

On day 2, replace medium with 5 mL of MnSC medium and return plate to incubator. See Troubleshooting 6.

On day 4, dissociate cells and plate 5 × 104 cells/well (6-well plate) containing iMEFs using hES medium as the final medium and return plate to incubator.

Change hES medium daily.

On day 14–21, the reprogrammed cells should now transform from fibroblast morphology to a round shaped morphology (Figure 6) and can be collected for characterization of pluripotency markers (Figure 7) and reprogramming efficiency and can be further analyzed for iPSC phenotype and pluripotent state characterization as needed.

Figure 6

Reprogramming Process

Bright-field representative images at different days after AOX15 transduction of MnSCs. Scale bar, 30 μm.

Figure 7

Reprogrammed iPSCs

Representative images of immunofluorescence analysis of markers indicative of pluripotency in control somatic MnSCs and AOX15 reprogrammed iPSCs. Scale bar, 30 μm, for all images.

If several colonies arise from one well during the reprogramming process, every single clone should be independently expanded for characterization and freezing.

Expected Outcomes

AOX15-iPSCs can be generated and passaged within 3–4 weeks. IPSC clones can be expanded for characterization. We recommend the following characterization assays: Immunocytochemistry for the visual detection of markers indicative of pluripotency, flow cytometry for a quantitative estimate of reprogramming efficiency (NANOG, OCT4, TRA-1-60, TRA-1-81, SSEA4, LIN28,). We expect over 80% of the cells to be positive for mentioned pluripotency markers. Additionally, qRT-PCR can be performed to confirm the expression of pluripotency genes (including OCT4, SOX2, hTERT, DNM3TB, KLF4, NANOG, ZFP42, among others) (Lopez-Caraballo et al., 2020).

Pluripotency is not a single state. Further characterization of the functional status of reprogrammed cells' pluripotency, including differentiation capacity, efficiency, and other molecular signatures can be performed (Liu et al., 2017; Lopez-Caraballo et al., 2020; Mishra et al., 2018). To this end, a list of recommended antibodies for immunocytochemistry is provided in the Key Resources Table.

We recommend dedicating at least three to four wells to each reprogramming quality control assay and isolating and characterizing at least three to four iPSC clones to allow pluripotency acquisition efficiency analysis and further pluripotent characteristics studies.

Quality Control: Measure of Viral Packaging

When performing the viral packaging of pMXs encoded reprogramming factors, it is important to measure the viral transduction efficiency using pMX-ASF1A and pMX-SOX15 bicistronic vectors that express GFP. Only viral supernatant reaching a concentration of 80% GFP positive cells with 1 mL supernatant (and over 95% with 3 mL) should be used for cell reprogramming.

These plasmids and flow cytometric analyses can also be used to estimate transfection efficiency and troubleshoot any problems with viral packaging.

Quality Control: Measure of Reprogramming Efficiency

Three to four weeks after MnSC transduction with reprogramming factors, the reprogrammed cells should transform from fibroblast morphology to a round shaped morphology. The cells can be passaged for characterization of iPSC phenotype and reprogramming efficiency using immunofluorescence labeling of pluripotent markers (NANOG, OCT4, TRA-1-60, TRA-1-81, SSEA4, LIN28). The original somatic MnSC should be used as negative control of expression and also to produce the reference short tandem repeats (STR) profile for the iPSC lines generated.

Limitations

Although the efficiency of AOX15 reprogramming has been significantly improved by using MnSCs instead of dermal fibroblasts (Lopez-Caraballo et al., 2020), iPSC generation efficiency remains relatively low and reprogramming rates vary from 10% to 0.0001% (Gonzalez-Munoz et al., 2014; Gonzalez-Munoz and Cibelli, 2018). The following limitations should be mentioned specifically. First, this protocol renders efficient reprogramming when using MnSCs; other cell types may require further optimization. Second, based on our experience low passage MnSCs (passage 3–6) should be used for reprogramming. Enzymatic dissociation or passaging and long-term culture has been described to affect the epigenetic state of the cell and to hinder efficient reprogramming (Halley-Stott and Gurdon, 2013; Kim et al., 2010; Streckfuss-Bomeke et al., 2012). Third, this approach depends on the quality and quantity of viruses, and as explained in this protocol, virus titration using 293T transduction should be done after each production and only highly efficient supernatants should be used.

Troubleshooting

Problem 1

MnSCs do not reach 80%–100% confluency one week after isolation (related to step 13)

Potential Solution

Replate cells into smaller plates to have a cell density above 104 cells/cm2

Problem 2

MnSCs do not proliferate properly (related to step 15)

Do not dilute cells below 5 × 103 cells/cm2. Low confluency gives rise to poor cell proliferation and early senescence. If cells do not reach confluency after 3–5 days of cell culture we recommend replating the cells into new dishes at higher cell density

Problem 3

MnSC sample population is not homogeneous (related to step 15)

The user may choose to perform cell sorting for the mesenchymal markers CD90, CD105, CD73, CD44 and CD13 positive cells (and negative for CD45, CD34, and HLA-DR). However, it is not necessary. Following this isolation protocol, more than 99% of isolated cells display the mentioned markers profile

Problem 4

Low 293T transfection efficiency (related to step 17)

Uneven distribution of cells may result in low 293T transfection and thus and thus virus titer. Rock the plate back and forth when seeding the cells. The user may check cell distribution under the microscope prior to incubation. Also, the user should carefully swirl the plate after adding each respective plasmid

Problem 5

Low virus titer (related to step 17)

Cells should reach 80%–90% confluency at the moment of transfection to ensure higher transfection efficiency and titer of viruses.

Problem 6

Excessive cell death after viral transduction (related to step 31)

Uneven distribution of cells may result in cell death after viral infection. Also, check the confluency of MnSCs at the moment of transduction, it should be over 50% for proper survival after transduction.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Elena Gonzalez-Munoz egonmu@uma.es.

Materials Availability

All material used are listed in Key Resources Table and any further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact.

Data and Code Availability

This protocol does not include the generation of datasets.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
goat anti-OCT4	Santa Cruz Biotechnology	Cat#sc-8628
rabbit anti-NANOG	Santa Cruz Biotechnology	Cat#sc-33760
rabbit anti-LIN-28	Santa Cruz Biotechnology	Cat#sc-67266
rabbit anti-SOX2	Abcam	Cat#AB5603
mouse anti-TRA-1-60	Chemicon/Millipore	Cat#MAB4360
mouse anti-SSEA4	Developmental Studies Hybridoma bank (Iowa)	Cat#MC-813-70
Alexa Fluor 488 donkey anti-mouse IgG (H+L)	Life Technologies	Cat#A21202
Alexa Fluor 488 donkey anti-rabbit IgG (H+L)	Life Technologies	Cat#A21206
Alexa Fluor 555 donkey anti-rabbit IgG (H+L)	Life Technologies	Cat#A31572
Alexa Fluor 555 donkey anti-mouse IgG (H+L)	Life Technologies	Cat#A31570
Alexa Fluor 488 donkey anti-goat IgG (H+L)	Life Technologies	Cat#A11055
Bacterial and Virus Strains
pMXs-GFP	Cell Biolabs	Cat#RTV-053
pMXs-hOCT4	(Takahashi et al., 2007)	Addgene Cat#17217
pMXs-hASF1A	(Gonzalez-Munoz et al., 2014)	Supplied after request
pMXs-SOX15	(Lopez-Caraballo et al., 2020)	Supplied after request
Gag/Pol Retroviral vector	(Reya et al., 2003)	Addgene Cat#14887
pCMV-VSV-G	(Stewart et al., 2003)	Addgene Cat#8454)
Chemicals, Peptides, and Recombinant Proteins
DMEM/F12	Thermo Fisher Scientific	Cat#11320-082
MEM Non-Essential Amino Acids Solution (100×)	Thermo Fisher Scientific	Cat#11140068
Opti-MEM I Reduced Serum Media	Thermo Fisher Scientific	Cat#31985062
L-Glutamine	Thermo Fisher Scientific	Cat# 21051024
Penicillin-Streptomycin	Thermo Fisher Scientific	Cat# 15070063
2-mercaptoethanol	Thermo Fisher Scientific	Cat# 21985023
Fetal Bovine Serum, Regular (Heat Inactivated)	Corning	Cat# 35-011-CV
KO-Serum Replacement	Thermo Fisher Scientific	Cat# 10828028
2-mercaptoethanol	Thermo Fisher Scientific	Cat# 21985023
Recombinant human basic FGF-premium grade	MACS-Miltenyi Biotec	Cat#130-093-843
0.05% Trypsin/EDTA	Thermo Fisher Scientific	Cat# 25300054
Mitomycin C from Streptomyces caespitosus	Sigma-Aldrich	Cat# M4287-2MG
Gelatin Solution	Sigma-Aldrich	Cat#G1393
Polybrene	Sigma-Aldrich	Cat#H9268-10G
Polyethylenimine, Linear, MW 25000, Transfection Grade (PEI 25K)	Polysciences	Cat#23966
Venor GeM Classic mycoplasm test	Minerva Biolabs	Cat#11-1050
Histopaque 1077	Sigma-Aldrich	Cat#10771-100ML
GlutaMAX Supplement	Gibco-Thermo Fisher	Cat# 35050061
Paraformaldehyde	Electron Microscopy Sciences	Cat#:15710
DPBS	Corning	Cat#:21-031-CM
Donkey Serum	Equitech-Bio	Cat#:SD30-0500
Triton X-100	Fisher Scientific	Cat#:BP151-500
Normal Donkey Serum	Sigma-Aldrich	Cat#D9663
Experimental Models: Cell Lines
Human: HEK293T/17 cells ATCC Cat# CRL	11268; RRID: CVCL_1926	Human: HEK293T/17 cells ATCC Cat# CRL
EmbryoMax Primary Mouse Embryonic Fibroblasts	Merk-Millipore	PMEF-CFL

5 μg Gag/Pol
3 μg VSV-G
7 μg pMX (transgene vector: pMX-ASF1A, pMX-SOX15 or pMX-OCT4)
500 μL of pre-warmedOptimem medium

1 mL pMX-SOX15	1 mL pMX-ASF1A	1 mL pMX-OCT4 negative Control
3 mL pMX-SOX15	3 mL pMX-ASF1A	3 mL pMX-OCT4 negative Control